Single Molecule Proteomics

ABSTRACT

This disclosure comprises devices and methods for determining the identity of individual protein molecules in a complex mixture by unfolding the protein into a polypeptide, tagging selected residues on the polypeptide with selected oligonucleotide sequence tags that recognize selected residues on said polypeptide, and then detecting the oligonucleotide sequence tags.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119 to U.S.provisional patent application, U.S. Ser. No. 62/029,376, filed Jul. 25,2014, entitled “SINGLE MOLECULE PROTEOMICS,” the entire contents ofwhich are incorporated herein by reference.

BACKGROUND OF DISCLOSURE

A number of methods are available for analyzing protein samples. Thisincludes immunoassays, microarrays, 2-D gel electrophoresis and massspectrometry. Recently, analysis of single protein molecules has beenexplored.

SUMMARY OF DISCLOSURE

Aspects of the disclosure relate to methods for identifying proteins,e.g., proteins present in a complex mixture. In some embodiments, themethods involve experimentally determining a distinct fingerprint foreach individual protein based on the location of specific residues alongthe polypeptide length of the protein. In some embodiments, the proteinis denatured/unfolded into a polypeptide form and the linear length ofthe polypeptide is determined and features along its length areanalyzed. In one embodiment the linear length of the polypeptide isanalyzed by stretching the polypeptide and determining the pattern orlocation of distinct residues along its length using an imaging orscanning method. The distinct residues can be determined by a number ofmeans including labeling them with detectable labels that are specificto particular residues or that are conjugated to other molecules, e.g.,antibodies, that are specific to particular residues. Alternatively, insome embodiments, the length of the polypeptide is analysed by passingit through a nanopore or nanogap and the pattern or location of distinctresidues is determined by recording the length of time between signals.In some embodiments, an experimentally determined location of distinctresidues in each individual polypeptide is then compared to a databasecomprising the location of distinct residues in each known or predictedprotein. It is estimated that there are approximately 20,000 proteins inthe human proteome as well as alternatively spliced isoforms, mutants,fusions, and versions modified by glycosylation, phosphorylation,methylation and acetylation, etc. In some embodiments, a match to thedatabase indicates the identity of the unknown protein in the complexmixture under analysis. In some embodiments, methods disclosed hereinare applied to complex protein mixtures from biological samples. In someembodiments the samples are of high complexity and dynamic range, e.g.,the blood proteome and in some embodiments, such as single human cellsor lower eukaryotes such as yeast the dynamic range is lower. In someembodiments, the aim is to access medium to low abundance proteins.Accordingly, some aspects of the disclosure involve the priorfractionation, enrichment and/or depletion of certain sub-sets of theprotein population.

In some embodiments, methods provided herein comprise analyzing thelinear length of a polymer to determine a pattern along a polymer (andoptionally the length of the polymer). In some embodiments, the methodsfurther comprise using the pattern along the polymer (and optionally thelength) to determine or assign the identity of the polymer or candidatesfor the identity of the polymer.

In other embodiments, methods provided herein comprise analyzing thelinear length of a polymer to determine the location, distance betweenand/or order of specific residues and optionally the length of thepolymer. In some embodiments, the methods further comprise using theorder, relative distance or coordinates of the specific residues on thepolymer (and optionally length) to determine or assign the identity ofthe polymer.

In other embodiments, methods provided herein comprise analyzing thelinear length of a polymer to determine the location, distance betweenand/or order of specific residues and optionally the length of thepolymer. In some embodiments, the methods further comprise using theorder, relative distance or coordinates of the specific residues on thepolymer (and optionally length) to determine or assign the identity ofthe polymer or candidates for the identity of the polymer. In someembodiments, only a subset of residues are analysed.

In other embodiments, methods provided herein comprises unfolding theprotein into a polypeptide. In some embodiments, the methods furthercomprise analyzing the linear length of the polypeptide to determine thelocation, distance between and/or order of specific residues andoptionally the length of the polypeptide. In some embodiments, themethods further comprise using the length and/or order, relativedistance or coordinates of the specific residues on the polypeptide (andoptionally the length of the polypeptide) to determine or assign theidentity of the protein or candidates for the identity of the protein.

In other embodiments, methods are provided herein that compriseunfolding a protein into a polypeptide, analyzing the linear length ofthe polypeptide to determine the location, distance between and/or orderof specific residues and optionally the length of the polypeptide, andusing the length and/or order, relative distance or coordinates of thespecific residues on the polypeptide (and optionally the length of thepolypeptide) to determine or assign the identity of the protein orcandidates for the identity of the protein. In some embodiments, only asubset of residues are analysed.

In other embodiments, methods are provided herein that compriseunfolding a protein into a polypeptide, and analyzing the linear lengthof the polypeptide to determine a pattern of a physicochemical property,e.g., hydrophobicity. In other embodiments, the methods further compriseusing the length and/or the pattern of a physicochemical property todetermine or assign the identity of the protein or candidates for theidentity of the protein.

In other embodiments, methods are provided herein that compriseunfolding a protein into a polypeptide and labeling residues along thepolypeptide. In some embodiments, the residues are labeled prior tounfolding. In some embodiments, the residues are labeled followingunfolding. In some embodiments, such methods further comprise analyzingthe linear length of the polypeptide to determine a distinct pattern oflabels and optionally the length of the polypeptide. In someembodiments, such methods further comprise using the distinct pattern oflabels on the polypeptide (and optionally the length of the polypeptide)to determine or assign the identity of the protein or candidates for theidentity of the protein.

In some embodiments, methods provided herein comprise unfolding aprotein into a polypeptide, labeling residues along the polypeptide,analyzing the linear length of the polypeptide to determine thelocation, distance between and/or order of the labeled residues andoptionally the length of the polypeptide, and using the length and/ororder, relative distance or coordinates of the labeled residues on thepolypeptide (and optionally) the length of the polypeptide to determineor assign the identity of the polypeptide or candidates for the identityof the protein.

In some embodiments, methods provided herein comprise depleting highabundance proteins and/or enriching medium and/or low abundanceproteins. In some embodiments, such methods further comprise analyzingthe remaining polypeptides at the single molecule level. In someembodiments, such methods further comprise comparing a pattern/data fromeach individual polypeptide to a protein database. In some embodiments,the comparison yields the identity of the protein or candidates for theidentity of the protein.

In some embodiments, the methods further comprise enriching specificproteins. In some embodiments, the methods comprise analyzing theenriched polypeptides at the single molecule level. In some embodiments,the methods further comprise comparing a pattern/data from eachindividual polypeptide to a protein database. In some embodiments, thecomparison yields the identity of the protein or candidates for theidentity of the protein.

In some embodiments, an experimentally derived pattern of labels or datafor label location distance between and/or order of, is compared to oneor more in silico generated patterns of known proteins or to thesequence of known proteins. In some embodiments the apparent length ofthe polypeptide is also used in making a determination. In other cases(where for example the protein may be truncated) the length is not used.Other features of the protein may be determined to facilitate matchingto the database, for example the net charge on the polypeptide may bedetermined.

Optionally more than one type of residue is labeled and, in some cases,each different type of residue is labeled with a distinct tag or label.In some embodiments, the tag is a DNA sequence. In some embodiments, theDNA tag acts as a docking site/handle for DNA PAINT (Points Accumulationfor Imaging in Nanoscale Topography) [Jungmann et al, Nano Lett. 2010Nov. 10; 10(11):4756-61.] In some embodiments, recording of the bindingof DNA PAINTS enables a super-resolution picture to be constructed.

In some embodiments, analyzing the linear length comprises translocatingthe polypeptide through a detection station (e.g., nanopore, nanogap)and making real time recordings of physical phenomena as each residuealong the polypeptide comes into the proximity of the station. In someembodiments, the physical phenomena is an optical signal. In otherembodiments the physical phenomena is an electrical signal. In someembodiments, both optical and electrical signals are analysed. In someembodiments, translocating of the polypeptide is controlled byelectrophoretic forces, hydrodynamic forces, pressure driven flow orphysical pulling.

In some embodiments the polypeptide passes through a nanopore and achange in ion flux is detected according to the label that passes thepore, as illustrated in FIG. 2A. In some embodiments, the polypeptidepasses through a nanogap electrode system. In some embodiments, thenanogap electrode system can produce a tunneling current and suchtunneling can be perturbed to a different degree by labels or tags onthe polypeptide, as illustrated in FIG. 2B. In some embodiments thenanogap is associated with an electrical field, capacitance,permittivity, etc. and a measurable quantity related to the electricalfield, permittivity, capacitance etc. is perturbed to different degreeby labels or tags.

In some embodiments, the polypeptide passes through an evanescent waveor a waveguide (e.g., a zero-mode waveguide). In some embodiments, thepolypeptide passes a fluorophore, whose fluorescence emission issensitive to its physical environment and different labels on thepolypeptide elicit different fluorescent responses such as attenuationof the signal. In some embodiments, the label on the residue is aquencher and quenches the signal of the label at the detection station.In some embodiments the label on the residue is a FRET acceptor and thelabel on the detection station is a FRET Donor or vice versa. In someembodiments, the label is polylabeled to elicit an enhanced response. Insome embodiments the station comprises features which enhance the signalof the label; such features include metallic structures at which areknown to enhance fluorescent or Raman signals. In some embodiments theproximal location to nanostructures is tuned.

In some embodiments, analyzing the linear length comprises passing thepolypeptide through a nanochannel or nanoslit and imaging the linearlength as it passes through. In some embodiments, analyzing the linearlength comprises attenuating the translocation of the polynucleotidethrough a nanochannel or nanoslit so that one or more images can betaken. In some embodiments the velocity of translocation of thepolypeptide is matched to the speed of read-out of the CCD chip(operated in Time-Delay Integration (TDI) mode) so that polypeptide canbe imaged whilst in motion; this can lead to faster data acquisition. Anarray of polypeptides can be stretched in an array of nanochannels. Insome embodiments, the polypeptide is placed on a surface in anon-globular form and preferably the polypeptide is stretched on thesurface. An array of polypeptides can be imaged using a CCD camera or a2-D array CMOS detector.

Various methods can be used to give a resolution beyond the diffractionlimit of light. Alternatively the surface can be scanned by methods suchas scanning probe microscopies (as illustrated in FIG. 2C) or laserscanning microscopies. In one embodiment, an optical signal is detectedas the labeled polypeptide passes through a nanopore or a nanogap.

In some embodiments, specific chemical moieties such as cholesterol areattached to the polypeptides. In some embodiments the residues that areattached comprise a polymer. Such a polymers may wrap around thepolypeptide to homogenize the backbone charge. Other polymers can begrafted onto one or both ends of the polypeptide. The polypeptide canthen be manipulated according to the properties of the polymer.

In some embodiments, an identity is assigned to each polypeptideaccording to the pattern of labels detected or data derived from thepolypeptide. Preferably the experimentally derived pattern is comparedto an in silico generated pattern or the experimentally derivedcoordinates are compared to the coordinates in the sequence of residuesof proteins in a database by using parallel computing. Such parallelcomputing includes use of clod-based computing and Graphics processingunit (GPU), which have a large number of processing cores.

In some embodiments, as an alternative to a comparison of predictedpatterns and data, experimental data is obtained of purified proteins, adatabase is created and then the test polypeptide is compared to thepreviously acquired experimental data.

In some embodiments the protein population is handled collectively untilthe detection step, whereupon each protein in the population is handledindividually and/or detected individually.

The abundance of each protein in the sample is determined by enumeratingthe number of occurrences of a match of individual polypeptides to eachprotein in a database.

In some embodiments, a whole process from sample collection to report ofresults can involve one or more of the following steps:

1) Collecting or acquiring a sample cells, tissues or organisms; in thecase of blood, preferably isolating plasma

2) Extracting or isolating proteins from the sample

3) Depleting high abundance proteins/Enriching lower abundance proteins

4) Labeling the proteins at one type of residue with a distinct label ortag

5) Optionally labeling the proteins at another type of residue using asecond distinct label or tag, which is distinguishable from the firstlabel or tag (and label further residues with distinct labels, ifnecessary)

6) Rendering the proteins into a substantially unfolded polypeptide form(this step can optionally occur before step 4)

7) Optionally contacting the polypeptide with moieties (includingpolymers such as spermine or polynucleotides) that facilitate physicalmanipulation of the polypeptide

8) Handling each polypeptide individually

9) Detecting the order, location or distance between labels or specificphysico-chemical features on the polypeptide or the distinct pattern oflabels or specific physicochemical features on the polypeptide;optionally filtering data using hardware/digital signal processing

10) Analyzing the experimentally derived pattern/data and assigning anidentity to the polypeptide under analysis wherein the analysis maycomprise comparison to a database of proteins and is preferablyconducted by parallel computing

11) Analysis is optionally done on the fly

12) Optionally providing a list of proteins present in the sample andoptionally their abundance in the sample

One of the simplest fingerprint parameter of a protein is whether itbinds to a specific probe or not, and this can be achieved using thedisclosure with medium to low abundance proteins by applying priorfractionation, enrichment and/or depletion. In some embodiments variousmeans of detecting proteins can be applied at the single molecule level,including binding to an antibody array.

Therefore, in some embodiments, the disclosure comprises:

(i) depleting high abundance proteins and/or enriching medium and/or lowabundance proteins or enriching specific proteins or purifying specificproteins; and

(ii) analyzing the enriched/purified/non-depleted proteins by singlemolecule detection

In some embodiments, the ultimate fingerprint of the proteins is theentire sequence of each polypeptide and this can be achieved using thedisclosure with medium to low abundance proteins by applying priorfractionation, enrichment and/or depletion.

Therefore, in some embodiments, the disclosure comprises.

(i) depleting high abundance proteins and/or enriching medium and/or lowabundance proteins or enriching specific proteins or purifying specificproteins;

(ii) analyzing the enriched/purified/non-depleted proteins, polypeptidesat the single molecule level;

(iii) detecting each individual residue or pairs of individual residuesor individual oligopeptides along the length of the polypeptide; and

(iv) processing the collected data to provide the complete or partialsequence of the target polypeptide(s). In some embodiments, a motorprotein/unfoldase/chaperonins are applied to the polypeptide(s) beingsequenced. In some embodiments, in which pairs of amino acids oroligopeptides are individually detected, the identity of the signals aredecoded with reference to a look up table and a sliding window ofanalysis is optionally applied. This analysis can include for example asliding window comprising a first unit as amino acids 1-8, 2-9, 2-10,4-11 etc. Eight amino acids is approximately the length of resolvabilityof the MspA nanopore which has a narrow constriction.

In some embodiments the proteins comprise the proteome of body fluidssuch as blood or enriched/depleted versions thereof; plasma is preferredover whole blood or serum due to lower ex vivo protein degradation. Theproteome of other body components or waste can also be examined. In someembodiments the proteins comprise the protein contents of a single cell.In some embodiments the proteins comprise secreted proteins frommultiple cells or a single cell. In some embodiments proteins compriseproteins that have recently been transported across a membrane and as aconsequence of the transporting process are rendered as unfoldedpolypeptides.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides a non-limiting illustration of a workflow for proteinfingerprinting; and

FIGS. 2A-2C provide non-limiting illustrations of labeled or unlabeledprotein fingerprinting.

DETAILED DESCRIPTION OF DISCLOSURE

Aspects of the disclosure relate unfolding of the protein and disposingit in a non-globular substantially linear manner so that its length andresidues along its length can be examined. Other aspects of thedisclosure relate labeling of the residues and/or the labeling of aprobe, which is optional depending on the mode of the disclosure beingpracticed. Other aspects of the disclosure relate to detection offeatures along the linear length of the polypeptide. Other aspects ofthe disclosure relate to identifying the protein by comparison of theexperimentally derived details or patterns to a database of the expecteddetails or patterns of known proteins. These and other aspects of thedisclosure are described further below. In some embodiments, methods areprovided herein that enable proteins to be analysed directly from acomplex mixture without the need for prior separation or purification.

Extracting Proteins

Cell-free Proteins can be extracted form body fluids using standardtechniques. Blood can be spun down, and the Plasma fraction is collectedand the protein fraction therein is precipitated and collected. Proteinscan be extracted from cells using detergents such CellLytic M(Sigma-Aldrich).

Single Cells

Single cells can be processed (including cultured) sorted and trapped inmicrofluidic devices. The trapped cells can lysed on-chip and thecontents of each individual can be collected and prepared separately,much as has been done for RNA and RNA in C1 system by Fluidigm.

Depletion, Normalization and Enrichment

The dynamic range of proteins in biological samples can extend over nineorders of magnitude. Certain high abundance proteins are at such highlevels that the low abundance proteins are very difficult to detect andeven medium abundance proteins are difficult to quantify. Therefore inorder to detect the low and medium abundance proteins specific measuresprovided by this disclosure need to be taken.

Fractionation

The protein population can be fractionated prior to analysis. Thisfractionation can be based on affinity purification of different classesof proteins, for example immunoglobulins can be isolated by binding totheir Fc portions.

One important embodiment of this disclosure is fractionation based onabundance and/or electrophoretic properties and/or other physicochemicalproperties, e.g., those detected by liquid chromatography, massspectrometry etc. In one specific embodiment, the population of proteinsis fractionated by using isoelectric focusing, e.g., in 1-D or 2-D gelelectrophoresis or via chromatography (iso-focusing chromatography).Then the bands pertaining to the high abundance proteins (bands withhigh intensity after staining) are cut out and removed. The remainingproteins in the gel can then be eluted out and analyzed by the methodsof this disclosure. Taking this to the extreme, all visible bands arecut out, and elution is carried out on the remaining parts of the gel,collecting proteins that were not visible. Of course low abundanceproteins that co-locate in the gel with the high abundance proteins willbe lost. The bands can be cut out by automated methods. Membraneproteins which are typically hydrophobic are hard to access by gelelectrophoresis as are alkaline proteins, therefore the subset ofproteins that are analysed are depleted of such proteins.

Depletion

In order to prevent high abundance proteins from drowning out lowabundance proteins, common high abundance proteins such as humanalbumin, transferrin, haptoglobin, a-1-antitrypsin, IgG, and IgA orothers which have little value as biomarkers or as components of thepathways of interest can be depleted from the protein population. Thereare a number of depletion kits available including, the ProteoPrep(Sigma) which in some embodiments of the disclosure are applied afterprotein extraction and preferably before unfolding into linearpolypeptide and before analysis.

Equalization of Protein Abundance

As an adjunct or alternative to depletion, the low abundance proteinsare enriched at the expense of high abundance proteins. There are anumber of enrichment kits available including the ProteoMiner (BioRad).The principle of the approach is the treatment of complex proteinsamples with a large, highly diverse library of oligopeptides (which aretypically attached to a support) whereupon each oligopeptide binds aunique recognition site on a protein. As the capacity of the library isfinite, the higher abundance proteins soon saturate the oligopeptidesspecies that they bind to and excess high abundance proteins are washedaway. In contrast, the lower abundance proteins do not completelysaturate the oligopeptides they bind to or if they do the excess amountof protein that is washed away is small compared to the case for highabundance proteins. The resultant effect is that the concentration ofthe different abundance classes are equalized to a substantial extentand the dynamic range of proteins in the sample is decreased.

Enrichment of Specific Proteins

Affinity ligands which bind to specific proteins of interest can beprovided. For example, the affinity ligands can target proteinsimplicated in cancer. After their capture, and the removal ofnon-captured proteins, the captured proteins can be released from theaffinity ligands and analysed by the methods of the disclosure. Thisenrichment can reveal whether a mutant, mis-spliced or truncated versionof a protein or a particular low abundance protein is present in apopulation.

The depletion and enrichment processes can be carried out multiple timesto enhance their effect.

In some embodiments of the disclosure the proteins aredepleted/enriched/equalized and then subject to sequencing of thepolypeptides.

In some embodiments of the disclosure the proteins aredepleted/enriched/equalized and then subject to fingerprinting andidentification of the polypeptides.

Microfluidic chips containing the principles above, linked with thepolypeptide analysis methods of the disclosure are part of thedisclosure. Fractionation, depletion, equalization, purification andenrichment can be integrated on chip. Preferably this involves,increasing internal surface area in the chip so that a greater amount ofthe capture reagents can be loaded onto the chip. The internal surfacecan be increased by including controlled pore glass or other membranousmaterial, beads or pillars/micro-posts inside the chip.

The advantage of microfluidic approach is integration, ease, increasedefficiency and reduction of contamination; depletion methods aremulti-step and usually lead to a significant keratin contamination. Alsoa multitude of polymeric contaminants are released from the plasticsused in off-chip methods that can lead to complication of the results.Much of this is avoided when the depletion, equalization or enrichmentsteps are carried out in the microfluidic device and especially whensubsequent analysis steps are integrated in the device. Enrichment,equalization or depletion reagents can be flowed across an array ofpillars to which they bind. The protein population is then flowedthrough and are able to bind to their specific ligands. In case of thedepletion approach, what does not bind and flows through, is subject tosubsequent steps of the disclosure such as unfolding and the analysis ofthe features and linear length of the polypeptides. In the case of theequalization and enrichment approach, what does not bind and flowsthrough initially is removed and the proteins that have bound are theneluted and subjected to the subsequent steps of the disclosure such asunfolding and the analysis of the linear length of the polypeptides.

All or some the steps of the disclosure, from sample loading, proteinextraction, labeling, unfolding, linearization to detection can becarried out in a lab-on-a-chip device.

Unfolding Proteins

Native proteins comprise tertiary and quaternary three-dimensionalstructures. Unfolding and refolding of proteins occurs routinely withincells.

In order to practice the methods of this disclosure, native proteinsmust first be unfolded into linear one-dimensional strings. This can bedone in a number of ways by heat, denaturants, and extreme pH. However,Nivala has described the use of an unfoldase, the chaperone protein,ClpX to unfold proteins to their polypeptide form so that they cantranslocate through a nanopore (Unfoldase-mediated protein translocationthrough an α-hemolysin nanopore. Nivala J, Marks D B, Akeson M. NatBiotechnol. 2013 March; 31(3):247-50). In addition to the approach usedby Nivala, it is disclosed herein that chaperone proteins or complexescan be added to a solution containing the proteins to unfold theproteins before they are analysed. This can be done under denaturingconditions so that the polypeptides remain substantially unfolded.Physical methods can be used as an alternative to enzymatic unfolding.This can include the application of heat (e.g., heat to above 75° C.)and/or a number of specific chemicals. The denaturing conditions (e.g.,using Urea, Formamide, SDS, GuCl) are compatible with of nanopore andnanochannel measurements depending on the concentration of reagentsused. Reducing agents can be used to break disulphide bonds whichcrosslink different segments of the polypeptide in the protein tertiarystructure. Suitable reagents include, TCEP, Dithiotrol (DTT), andbeta-mercaptoethanol (BME). Detergents such as SDS can induce unfolding;the addition of cation (e.g., NaCl) can increase the rate of unfolding.Chaotrophic agents can be used to unfold proteins. Guanimium chloride isone such effective reagent. Urea (8M at 60° C.) can also be used tounfold proteins.

Molecular Combing, Hydrodynamic Stretching, Electro-Stretching andMolecular Threading

The protein can be stretched on a surface by binding the N terminus or Cterminus to the surface. The termini can bind a defect on the surface.Preferably the surface is suitably derivatized and the polypeptide bindsto a specific chemical group.

-   -   Alternatively, one can take advantage of a common set of        residues present in a plurality of polypeptides. Many proteins        contain a common signal peptide or leader sequence (typically a        short 5-30 amino acid length of the N-terminus), which can        facilitate transport of the protein. Often the signal peptide        comprises a stretch of hydrophobic amino acids that tend to form        an alpha-helix. In addition, frequently, signal peptides have a        short positively charged stretch of amino acids.

Such leader sequence can be targeted for capture by an antibodyrecognizing said leader sequence, so that proteins containing the leadersequence can be captured by their leader sequence. Alternatively, in thecase of recombinant proteins a leader sequence or tag (e.g., histidine,FLAG-peptide) can be engineered onto the polypeptide; multiple copies ofthe leader can be included. The leader can then bind to its ligandattached to the surface (e.g., a copper coated surface can being tohistidine). Alternatively, a leader sequence can be conjugated onto theend of all polypeptides by reaction with the N or C terminus. Anartificial leader sequence can comprise an unstructured polyanionicsequence such as 65-amino-acid-long glycine/serine tail including 13interspersed negatively charged aspartate residues. Capture can be onthe basis of hydrophobicity (e.g., by contact with alkyl thiols coatinga gold surface) or charge (e.g., by contacting positively chargedPoly-L-lysine coated surface). Binding to such natural leader sequencescan be a way of enriching proteins destined to be secreted. A repertoireof different polypeptide binding reagents can be patterned on a surfaceso that most all different types of polypeptides can be captured.

As an alternative to a leader sequence one or more biotins can beengineered in vitro or in vivo on to the ends of the polypeptidesallowing the polypeptide to be captured on a streptavidin or anti-biotinantibody coated surface.

The surface can be patterned with multiple capture reagents. Forexample, stripes can be made containing hydrophobic or hydrophilicresidues, or residues with negative or positive charges. Polypeptideswith different leader sequences can then be immobilized to differentlocations on the surface.

One of the stretching methods described below can then be applied to thecaptured polypeptides.

The polypeptide can be combed onto a surface by a using a recedingmensicus approach. To achieve the receding meniscus a droplet containingthe polypeptides may be translated across a surface, or a dropletcontaining the polypeptides can be allowed to dry on a surface or asurface can be pulled out of a trough containing a solution containingthe polypeptides. Molecular combing has been applied to the stretchingof the Titin (TTN) polypeptide [Tskhovrebova L; Trinick J Flexibilityand extensibility in the titin molecule: Analysis of electron microscopedata J MOL BIOL 310 755-771, 2001].

Molecular threading involves dipping a needle into a solution containingthe polymer of interest, allowing a single polymer to attach to the tipof the needle and then passing the needle over a surface to deposit andstretch the polymer on the surface. This has been achieved forpolynucleotides and can be extended to polypeptides.

Polypeptides can also be stretched by application of an electric field.Preferably one end of the polypeptide is first attached to a surface.

The polypeptides, once attached at one terminus, can be stretched byfluid flow. The polypeptides can then be imaged whilst dangling in thefluid flow. Alternatively, the polypeptides can be allowed to settle onthe surface; providing suitable chemical attachment points on thesurface can facilitate this. For example, some polypeptides can bind toa negatively charged Mica surface.

As an alternative to stretching polypeptides on the plane of thesurface, they can be stretched perpendicular to the surface. This can bedone by attaching a bead to one end of the polypeptide and stretchingthe polypeptide upwards using magnetic tweezers. Confocal microscopy orlight sheet microscopy can then, for example, be is used to define thelocations of labels along the polypeptides.

All the DNA stretching methods that involve the attachment of a DNA endto a surface can be performed on a large number of molecules inparallel. One molecule can be prevented from overlapping with another bytailoring the concentration of polypeptides in the solution from whichthey are deposited. In the case of molecular threading, a comb-likestructure can be used to deposit a plurality of polypeptides inparallel, at predestined separations.

Stretching by Nanoconfinement

As with polynucleotides, polypeptides can be stretched bynanoconfinement, in nanochannels, nanogrooves or nanoslits. Thestretching can be facilitated or enhanced by using hydrodynamic flow incombination with nanoconfiment.

Several polypeptides can be stretched, head-to-toe or toe-to-head in asingle nanochannel but the dimensions of the nanochannel and the gap intime between entry into the nanochannel will ensure in the majority ofcases enough of a gap, that the start of one protein can bedifferentiated from the end of another. Nevertheless in someembodiments, the start and/or end of the proteins are tagged, preferablywith a label that can be differentiated from the labels along the lengthof the molecule.

Translocating Through a Nanopore

When proteins are unfolded they can be translocated through nanopores(Oukhaled et al Physics Review Letters, 98: 158101). To control thetranslocation Nivala et al (WO 2013123379) describe controlled unfoldingand translocation of proteins through the a-hemolysin (a-HL) pore usingthe AAA+ unfoldase ClpX. Nature biotechnology, 31: 247 (2013).

ClpX is a component of the ClpXP proteasome-like complex that isresponsible for the targeted degradation of numerous protein substratesin Escherichia coli and other organisms. ClpX forms a homohexameric ringthat uses ATP hydrolysis to unfold and translocate proteins through itscentral pore and into a proteolytic chamber (ClpP) for degradation. ClpXgenerates sufficient mechanical force (˜20 pN) to denature stableprotein folds, and because it translocates along proteins at a ratesuitable for primary sequence analysis by nanopore sensors (up to 80amino acids per second).

Natural or artificial leader sequences at the polypeptide terminus canbe deployed to help the polypeptide be attracted to the pore and/or tobe threaded through. An ssrA tag can be added for this purpose. ThisssrA peptide tag allows ClpX to specifically bind to the C terminus ofthe protein when it threaded through the pore into the trans compartment

Reversing Translocation and Repeating Measurement

In order to obtain a better accuracy in determining the characteristicsof the polypeptide, measurements can be repeated on an individualpolypeptide. This can be done by reversing the direction oftranslocation, for example, by switching the polarity of the electricfield. This can be done while a polypeptide is in the nanopore.Alternatively, immediately after the polypeptide has come all the waythrough the nanopores the polarity is reversed, providing a very highlikelihood that the same polypeptide will be threaded back in andtranslocated through. During the reverse translocation, measurements canbe made. The characteristics of the measurements may differ in theforward and reverse directions, especially if the pore has an asymmetricstructure from the trans to the cis side of the membrane.

Threading Polypeptides into Nanopores and Nanochannels

Proteins are heterogeneous, bearing different net charges and differentpolypeptides have different charges along their length. It ischallenging to thread a polypeptide molecule into a nanochannel ornanopores. However the threading can be facilitated by attaching aleader sequence to one or both ends of the polypeptide. When a pore isformed in a lipid bilayer, a cholesterol tag can be used to bring thepolypeptide to the lipid bilayer membrane. A polynucleotide sequence,which has a homogenous backbone charge can be added to end of thepolypeptide to facilitate its threading into the pore. In addition anarray of pillars/microposts or other structures can be placed adjacentto the nanochannel or nanopore/nanogap to guide the polypeptide to theorifice and facilitate threading.

Molecular motors can be used to pull the polypeptide through a porefollowed by comparison of the order of lysine and cysteine amino acidsto a reference. In some embodiments, it is possible to identify aprotein by detecting the order of ˜10-25 amino acid residues (e.g.,lysine and cysteine amino acid residues) in a single protein.

DNA can be translocated through microchannels, nanochannels andnanopores by pressure driven flow or by electrophoretic flow. Unlikepolynucleotides, polypeptides, due to their 20 amino acids, with variouscharges, have a heterogeneous charge pattern along their length, whichmakes electrophoretic translocation less straightforward than for theDNA case. Polypeptides can however be translocated using pressure, whichacts independently of charge. Nevertheless, much like traditionalelectrophoresis of proteins, the polypeptides can be contacted withreagents that neutralize charges, i.e. Sodium Dodecyl Sulphate (SDS).

For proteins, sodium dodecyl sulfate (SDS) is an anionic detergentapplied to protein sample to denature secondary and non-disulfide linkedtertiary structures, leading to linearized polypeptides and imparts anegative charge to the polypeptide.

Heating of a protein or a protein mixture in the presence of SDS canlead to a substantially unfolded state permitting binding of SDSthroughout the length of the polypeptide. Once SDS has been bound, thecharacteristic pI values of the proteins is no longer relevant; theprotein takes on a negative charge, and each protein has essentially thesame charge to mass ratio.

Proteins that have a greater hydrophobicity such as many membraneproteins, and those that interact with surfactants in their naturalmilieu, are harder to treat using SDS.

Nevertheless SDS can enable the polypeptide to be treated similarly to apolynucleotide and the stretching and combing and nanopores methodsdeveloped for DNA polymers can be applied to and optimized forpolypeptides. DTT or other reducing agents can break disulphide bonds,allowing proteins to fully unfold. Such a negatively charged peptidechain would go through a nanopore like a uniformly charged DNA molecule.

One problem is that protein pores can become unstable under certainprotein denaturing conditions however, solid-state pores have no suchproblem. However, the creation of bubbles with addition of SDS makesnanopores measurements somewhat difficult. However, anti-foamingreagents can be added to reduce this problem.

SDS and DTT can be combined, to unfold proteins and to allow them to bestretched on surfaces, in flows, in nanoconfinement and be transportedthrough nanopores.

Block Copolymer

One way to practice the disclosure is to fuse the polypeptide with aco-polymer (e.g., polynucleotide) that can be stretched by molecularcombing, molecular threading, fluid flow, confinement or by applying anelectric field. The stretching of the co-polymer enables the polypeptideto be co-stretched. A polynucleotide sequence of sufficient length tostretch on a surface is covalently linked to the C tor N terminus of apolypeptide. In one embodiment, the interaction with the surface is withone of the polypeptide ends and the stretching of the polynucleotideportion of the polymer causes the polypeptide portion to also stretch.Alternatively, both ends of the polypeptide can be linked topolynucleotides. Then the polynucleotide on one side attaches to thesurface.

Polypyrole or other conjugated polymers such as polyaniline,poly(ethylenedioxythiophene) can be conjugated to the ends of thepolypeptide to provide additional functionality.

Labeling

Protein/polypeptide labeling by chemical means involves the covalentattachment of labels/tags to amino acids using labels conjugated toreactive chemical groups that react with specific amino acid residues.There are a number of functional groups on proteins/polypeptides thatare available for labeling for the purposes of this disclosure. Thisincludes the following common types of functional groups: Primary amines(—NH2) which exists in lysine side chains and at the N-terminus;Carboxyls (—COGH) which are found in aspartic acid, glutamic acid sidechains and at the C-terminus; Sulfhydrils (—SH) which are present in theside chain of cysteines; Carbonyls (—CHO) which are created by oxidizingcarbohydrate groups in glycoproteins.

Labeling one type of residue is sufficient to fingerprint a protein andto identify it. Optionally more than one type of residue is labeled andpreferably each different type of residue is labeled with a distinct tagor label.

In a substantial number of cases the efficiency of labeling may notreach 100% but a sufficient number of labels are achieved per moleculeto identify the molecule.

Sypro Ruby and other protein stains can be used to label the polypeptidebackbone or certain classes of amino acids such as the basic amino acidsin the polypeptide. This helps to visualize the polypeptide. Without abackbone stain the correlation of labeled residues along a traceableline is adequate to visualize the polypeptide. Lysine residues can belabeled by NHS-ester chemistry. Cysteines can be labeled by maleimidechemistry. Histidines can be labeled by binding to metals such as Nickeland Copper. The N- and C-termini can also be labeled. Both cysteine andmaleimide chemistry can be used to label a polypeptide, one appliedafter the other.

Detection Nanopore-Mediated Detection

Solid-state, biological or hybrid nanopores can be used for detection.When the polypeptide enters the pore, a blockade in ionic current isdetected. Then when the first label passes the pore a further increasein blockade is detected. When the label has passed the pore the blockadeis decreased to the level of the polypeptide alone until the next labelis detected. If different labels are used then different degrees orduration of blockade are detected.

An advantage of biological nanopores or pores with some chemical groupsattached is that specific functionalities can be engineered into oradjacent to the pore. For example, a molecular motor protein can beattached to facilitate translocation of the polymer. Conjugated polymerscan be attached to the biological nanopores (e.g., a DNA nanopore) toprovide light emitting or light harvesting capability at the pore. Alight emitting capability at the pore leads to on-chip illuminationwithout the need for a separate light source. Similarly, a stain orintercalating dye can be added to a pore comprising DNA origami ornanostructure, which emit light at a higher wavelength than that atwhich they are excited. Similarly, fluorophores, chemically orbiologically coated fluorescent nanoparticles such as Quantum Dots(Invitrogen, Carlsbad) can be conjugated to biological pores.

Microscopy-Related Detection

Optical imaging and scanning methods can also be used for detection.Typically, the labels should be fluorescent dyes, particles or otherstructures or light-scattering particles. A CCD or CMOS chip can be usedto obtain a wide-field image of an array of polypeptides stretched on asurface or in nanochannels. A particular advantage of detectingpolypeptides on surface is that billions of molecules can be stretchedon a surface and then detected using fast imaging methods such as theTDI mode. This then is compatible with the dynamic range of proteinsthat might be encountered. Hence, using such surface-based stretching,even rare or low abundance can be detected with no or little depletion,equalization or enrichment.

Because polypeptides are generally short (e.g., by comparison to apolynucleotide) many useful sites of labeling are likely to be too closetogether to be resolved by optical microscopy. For this reason it isimportant to use super-resolution or high spatial resolution detectionmethods. A label carrying a DNA tag onto which DNA PAINTS can dockallows super-resolution imaging to be conducted. Different tags onlabels targeting different amino acids, enables multi-colorsuper-resolution imaging to be obtained.

In scanning optical approaches STED or SNOM microscopy can be used toobtain resolution beyond the diffraction limit of light. A coursegrained image is first obtained to locate the polypeptides on thesurface and then the path of the STED beams can be directed over each ofthe polypeptides. When the polypeptides are stretched or elongated innanochannels, then the STED beams can traverse along the path of thenanochannels. The nanochannels can be organized at predeterminedlocations with respect to their setting into an insert on the microscopeor with respect to an easily detectable marker on the substrate.

In some embodiments signal enhancement is achieved by proximity to ametal or by plasmonic effects, including those achieved by usingplasmonic structures such as a bow-tie or bulls eye.

Non-optical surface imaging or scanning methods can also be used. Thisincludes the electron microscopies (e.g. Transmission Electronmicroscopy, Scanning Electron microscopy) and the Scanning probemicroscopies (e.g., Scanning Tunneling Microscopy, Atomic ForceMicroscopy, Scanning Ion Conductance Microscopy). Providing a label withsome size larger than the polypeptide width or a shape is sufficient.The electron microscopies benefit from labels containing heavy metals ornanoparticles.

Determining the Pattern of Labels

In one way of practicing the disclosure, the location, distance betweenand/or order of labels is used to assign an identity to the protein. Inanother embodiment, a pattern of labels is used to assign the identity.The pattern may not allow one to determine the exact location, distancebetween or the order of labels. For example, with the resolutionavailable it may not be possible to tell which color label comes first,when the labels are substantially co-localised or a run of the samelabels that cannot be resolved may cover a portion of the polypeptide.

The rendition of labels along each polypeptide in the database is doneare blurred to the extent of the optical resolution. So if the opticalresolution is 250 nm, any residues that are labeled within a range of250 nm are blurred into one dot.

Determining the Pattern of a Physico-Chemical Property

The polypeptide can be analyzed to determine a pattern of aphysicochemical property of the amino acids along its linear length.This property can be the hydrophobicity of an amino acid, the charge onthe amino acid etc. The physiochemical property cab be determined by theinteraction of the amino acid side chains with a probe. The probe can bean AFM tip, its material composition or coated with a suitable chemicalgroups or biochemical residues. The probe can also be the residuesinside the lumen of a biological nanopore. This can be the nativeinternal nature of the lumen of a wild type pore, for example theAnthrax Toxin Pore has hydrophobic residues in a circular arrangementaround its internal diameter [A Phenylalanine Clamp Catalyzes ProteinTranslocation Through the Anthrax Toxin Pore. Science 29 Jul. 2005: vol.309 no. 5735 777-781]. The very hydrophobic amino acids comprise:valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, andcysteine.

The pore can also be engineered to present a specific physicochemicalproperty. For example, a cyclodextrin (or its derivatives) can beinserted into a nanopore to make the interior of the pore hydrophobic.When hydrophobic residues interact with the hydrophobic side chains, inan aqueous environment, the translocation of the polypeptide through thepore can show patterns of stalling. This leads to a characteristicpattern for a given protein, as each protein has a somewhat uniquepattern of hydrophobic residues.

Other intrinsic features of the polypeptide that can be analysed includeits electrostatic properties, adhesive properties, local folded state(see below), backbone flexibility (e.g., increased flexibility shows upas increased noise in a nanopores trace), elasticity, mechanicalstability, stickiness, fluorescence (see below), absorbance, and bindingaffinity to ligands.

A number of physico-chemical properties of the polypeptides can bedetermined by conducting Force-Distance curves using an Atomic ForceMicroscope (AFM). The AFM tip approaches and withdraws from the sampleon a pixel by pixel basis and measures interaction forces of the tipwith the polypeptide. The pixel sizes can be below 1 nm and thepositional accuracy can be 0.2 nm. AFMs are capable of performing largearrays of Force-Distance curves and means for this is integrated intothe software available form leading AFM vendors (e.g., Bruker). An arrayof Force-Distance curves enables multi-parametric imaging whichincludes: topographic, deformation, energy dissipation, elasticity,adhesion etc. information. Different regions of the polypeptide,comprising one or more amino acids, will give different responses in theforce curves. These can be displayed as a heat map across thepolypeptide and each polypeptide will have a unique heat map for atleast one of the physico-chemical properties determined. The unique mapof the physico-chemical property can be compared to the database ofpolypeptide containing experimentally derived or calculated patterns toprovide likelihoods for matches between the analysed polypeptide and thepolypeptides in the database. A combination of maps of differentphysico-chemical properties can be used to make the match. The AFM tipcan be composed of different material and can be coated with material(e.g., an antibody, a thiol group) that will give a particular characterto the physico-chemical measurement obtained. For example, a thiolcoated tip will interact with cysteine residues to give an increasedadhesion.

It is not necessary to know the basis of the physico-chemical phenomenabeing detected.

Determining the Pattern of Local Secondary Structure

Under particular denaturation conditions some types of local secondarystructure will remain in a polypeptide, and can be used as a way ofidentifying a particular protein, without or in addition to labelingspecific residues. Such local secondary structure can be detected byscanning probe microscopies or nanopores detection, for example. Theexperimentally derived database will, in this case, comprise proteinssubstantially treated and tested under the same conditions as thepolypeptide being tested.

Determining the Pattern of Intrinsic Fluorescence

Three amino acid residues, tryptophan tyrosine and phenylalanine areintrinsically fluorescent. However, they are neither bright norphotostable enough for standard single-molecule measurements. However,their fluorescence can be enhanced by proximity to a metal. Polypeptidesare translocated through a nanopores or nanochannel designed withintegrated metallic structures to enhance fluorescence; the enhancedfluorescence occurs at a detection station such as a nanopore, on asurface or in a nanochannel/nanoslit.

Determining the Location of Labels or Distance Between Two or MoreLabels

There are two ways that the location of labels can be determined. One isby precise coordinates of each label in relation to the start and end ofthe detectable length of the polypeptide. The length and coordinates ofthe label can be determined by making physical measurements of distance.For example, this can be done by using a calibrated optical microscopeor AFM. If the ends of the polypeptide are not precisely determined, forexample if the ends have curled up, are not fully stretched or remainglobular, then the distance between labels can be determined. The sameprinciples can be applied to the detection of the location of ordistance between particularly physico-chemical properties.

Alternatively the length can be determined by using time oftranslocation through a nanopore as a proxy. For example, when thepolypeptide enters the nanopore an increase in blockade in ionic currentcan be detected. When the polypeptide leaves the pore a decrease inblockage is detected. How the time of translocation relates to distancesor lengths can be calibrated by using one or more standards. When alabel on the polypeptide passes the pore, a characteristic change in theion blockade can be detected, e.g., a further increase in blockade isdetected while the part of the polypeptide bearing the label is in thenanopore. If the label stalls or slows down the translocation of thepolypeptide, the blockade event is longer lived. The temporal occurrenceof the increased blockade and its duration in relation to the blockadeassociated with the translocation of the polypeptide is recorded. Ifthere are multiple labels multiple blockades will be detected. This sameprinciple of changes in extent and duration of ion blockade is appliedwhen physico-chemical properties rather than labels are detected. Apattern of ionic flux changes (e.g., blockades) are determined for thelength of the polypeptide and can be examined to reveal the location oflabels in relation to the start and end of the polypeptide. If theblockade corresponding to the ends of the polypeptide are not recordedor not used, the time between consecutive blockades due to the labelscan be detected.

Determining the Relative Order of Two or More Labels

When two or more labels are used the location of or distance betweenlabels need not be determined, just determining the relative order ofthe different labels is sufficient to define a specific signature for aparticular polypeptide in order to determine its identity.

Comparison to a Database

An identity can be assigned to a protein, without necessarily carryingout a comparison to a database. In some case it is sufficient to saythat a given protein is distinct from another protein based on itspattern. However in other cases a comparison is made against a databaseto determine if a match to the protein exists in the database and if itdoes, what protein in the database it corresponds to. If the patterncorresponds to the expected pattern of a protein in the database (theprimary sequence of such proteins should be substantially known) then itis reasonable to assume that the experimentally derived pattern is forthe same protein (or at least a closely related protein) as that matchedin the database. This comparison can be made in a number of ways (or acombination of ways). The first is just by pattern matching. Thefollowing three are by obtaining specific types of data. The second isby comparing the order of labels on the polypeptide to the expectedorder of labels of candidate proteins in the database. The third is byusing the reasonably precise coordinates of the label on the polypeptideand the fourth is to use the reasonably precisely determined distancebetween each of the labels on the polypeptide to compare against adatabase of proteins containing such data. The experimentally deriveddata is normalized to take into account the physical rendering of thepolypeptide. For example the extent of unfolding or stretching willdetermine the distance between labels or the rate of translocation ofthe polypeptide will determine the distance between labels (using timeas a proxy for distance). This normalization can be done against aspiked-in known polypeptide (or other polymer) with known distancesbetween labels. As an alternative to such normalization, a number ofdifferent stretching factors can be computationally applied to theexperimentally derived patterns or data and then a comparison at eachstretching factor can be done against the database. A match to thedatabase at a particular arbitrarily assigned stretching (even if theactual stretching factor is not determined) can be sufficient to make adetermination of the identity of the protein. To allow for spliceisoforms and mutant proteins, the complete pattern may not match thedatabase but if a substantial part matches, especially over a contiguousstretch, then the identity of the protein is assigned to the match withthe caveat that the protein may be an isoform or a mutant. In some casesthe protein will be a fusion protein. In this case, part of theexperimentally derived data or pattern for a single polypeptide willmatch one protein in the database and another part of the experimentallyderived data or pattern will match another protein (or no protein) inthe database. Usually the match to each of the proteins in the databasewill only be over part of the length of the protein in the database. Insome cases the pattern or data from a polypeptide will be partialbecause the protein may be truncated or it may have broken duringhandling.

In most cases, the experimentally derived pattern is compared to an insilico generated pattern by using parallel computing. For example, theanalysis can be run using a graphics card (GPU) on a desktop or laptopcomputer. Here the matching problem can be broken into segments and eachsegment is run on a different core of the GPU. One way to do this is toassign an equal fraction of the database to each of the cores and thento run the comparison with the experimentally derived data or pattern ineach of the cores, in parallel.

The database can be constructed by gathering the sequence data of acomplete or partial set of proteins and performing computationalanalysis on each protein to access its length, the location of residuesalong its length and entering it into a column linked to the proteinname or accession number.

As an alternative, the database comprises or in addition comprises,entries derived from experimental data/patterns rather than expecteddata or predicted patterns. These entries are derived from the prioranalysis of individual proteins, which have been tested in a purified orsubstantially purified form. For example, a recombinant protein isexpressed and purified and then treated according to the experimentalembodiments of the disclosure and a pattern of labels or a datacomprising location of labels, distance between labels or order oflabels of different varieties are obtained and deposited in thedatabase. A polypeptide under analysis is then compared to the obtaineddatabase comprising of experimentally derived data and/or in silicocalculated data. If a particular polypeptide is not found in thedatabase, its pattern or data can be added to the database. Then furtheroccurrences of the polypeptide can be matched to the database.

Read Depth and Throughput

In order to access the least abundant or rarest proteins, the number ofproteins that must be analysed is huge. A large array of nanopores(e.g., ˜1 million) or fast microscopy based approaches are needed. Alsoto achieve sufficient throughput with nanopore methods, as fast a speedof translocation that can borne by the detection system should be used.This necessitates the avoidance of motor proteins and chaperonins suchas ClpX and prefers fast translocation using electrophoretic forces forexample, at a speed that does not provide resolution of every aminoacid. Not achieving resolution of every amino acid is compatible withdetection of the occurrences of cysteine and/or lysines, for example theoccurrence of 1 out of the 20 or 2 out of the 20 different amino acidswill be less frequent and require lower resolution to be detected.

With optical approaches, Time-Delay Integration (TDI) based imaging ispreferred. Super-resolution optical, scanning probe or electronmicroscopies allow a greater density of molecules to be imaged per area.However, to achieve high throughput super-resolution, optical imagingmethods those that require the imaging of a field of view over multipleframes should be preferably avoided. Methods that utilize the previouslycharacterized point spread function of known fluorescent labels tocalculate an image in super-resolution are preferred. Of the non-opticalapproaches electron microscopy is preferred because an image of a largenumber of molecules can be obtained using a 2-D detector such as a CCDcamera. It is preferred that time-delayed integration CCD imaging isused where stage movements are coupled with chip read-off. High-speedSPM can also be used.

One advantage of mapping specific residues, is that there is a massivespeed-up compared to when all residues have to be detected (anddiscriminated). In most cases only a pattern or the order of labels orapproximate location or distance between residues is needed. In thesecases objective magnification as low as 40× and 20× can be used(preferable with high numerical aperture optics, such as Zeiss 20× 0.8NAor Nikon 20× 0.75 NA). When this is coupled with TDI imaging with alarge chip a very large number of molecules can be imaged in a shortspace of time. The limit to the speed can become the rate at which thedata is transferred off the CCD chip to the computer. This data transferrate will improve over time.

Alternative Embodiments

The methods described above can be extended beyond polypeptides. In someembodiments a polynucleotide passes a fluorophore, whose fluorescenceemission is sensitive to its physical environment and different bases,base sequence motifs, 2mers, 3mer, 4mers, 5mers, 6mers or labels thereonelicit different fluorescent responses such as attenuation of thesignal. In some embodiments, the label on the residue is a quencher andquenches the signal of the label at the detection station. In relatedembodiment one or more contiguous bases directly elicit some change inan optical property at the detection station. In some embodiments thelabel on the one or more base is a FRET acceptor and the label on thedetection station is a FRET Donor or vice versa.

Computer Implementations

It should be appreciated that methods disclosed herein may beimplemented in any of numerous ways. For example, certain embodimentsmay be implemented using hardware, software or a combination thereof.When implemented in software, the software code can be executed on anysuitable processor or collection of processors, whether provided in asingle computer or distributed among multiple computers. Such processorsmay be implemented as integrated circuits, with one or more processorsin an integrated circuit component. Though, a processor may beimplemented using circuitry in any suitable format.

Further, it should be appreciated that a computer may be embodied in anyof a number of forms, such as a rack-mounted computer, a desktopcomputer, a laptop computer, or a tablet computer. Additionally, acomputer may be embedded in a device not generally regarded as acomputer but with suitable processing capabilities, including a smartphone, tablet, or any other suitable portable or fixed electronicdevice.

Also, a computer may have one or more input and output devices. Thesedevices can be used, among other things, to present a user interface.Examples of output devices that can be used to provide a user interfaceinclude printers or display screens for visual presentation of outputand speakers or other sound generating devices for audible presentationof output. Examples of input devices that can be used for a userinterface include keyboards, and pointing devices, such as mice, touchpads, and digitizing tablets.

Such computers may be interconnected by one or more networks in anysuitable form, including as a local area network or a wide area network,such as an enterprise network or the Internet. Such networks may bebased on any suitable technology and may operate according to anysuitable protocol and may include wireless networks, wired networks orfiber optic networks.

Also, the various methods or processes outlined herein may be coded assoftware that is executable on one or more processors that employ anyone of a variety of operating systems or platforms. Additionally, suchsoftware may be written using any of a number of suitable programminglanguages and/or programming or scripting tools (e.g., MATLAB), and alsomay be compiled as executable machine language code or intermediate codethat is executed on a framework or virtual machine.

In this respect, aspects of the disclosure may be embodied as a computerreadable medium (or multiple computer readable media) (e.g., a computermemory, one or more floppy discs, compact discs (CD), optical discs,digital video disks (DVD), magnetic tapes, flash memories, circuitconfigurations in Field Programmable Gate Arrays or other semiconductordevices, or other non-transitory, tangible computer storage medium)encoded with information (e.g., protein fingerprint or sequenceinformation) and/or one or more programs that, when executed on one ormore computers or other processors, perform methods that implement thevarious embodiments of the disclosure discussed above. The computerreadable medium or media can be transportable, such that the program orprograms stored thereon can be loaded onto one or more differentcomputers or other processors to implement various aspects of thepresent disclosure as discussed above. As used herein, the term“non-transitory computer-readable storage medium” encompasses only acomputer-readable medium that can be considered to be a manufacture(e.g., article of manufacture) or a machine.

The terms “program” or “software” are used herein in a generic sense torefer to any type of computer code or set of computer-executableinstructions that can be employed to program a computer or otherprocessor to implement various aspects of the present disclosure asdiscussed above. Additionally, it should be appreciated that accordingto one aspect of this embodiment, one or more computer programs thatwhen executed perform methods of the present disclosure need not resideon a single computer or processor, but may be distributed in a modularfashion amongst a number of different computers or processors toimplement various aspects of the present disclosure.

As used herein, the term “database” generally refers to a collection ofdata arranged for ease and speed of search and retrieval. Further, adatabase typically comprises logical and physical data structures. Thoseskilled in the art will recognize methods described herein may be usedwith any type of database including a relational database, anobject-relational database and an XML-based database, where XML standsfor “eXtensible-Markup-Language”. For example, protein fingerprint orsequence information may be stored in and retrieved from a database.

Computer-executable instructions may be in many forms, such as programmodules, executed by one or more computers or other devices. Generally,program modules include routines, programs, objects, components, datastructures, etc. that perform particular tasks (e.g., tasks relating toFeedback control) or implement particular abstract data types.

Typically the functionality of the program modules may be combined ordistributed as desired in various embodiments.

Examples

An example workflow for protein fingerprinting and analysis is providedin FIG. 1. Supporting details and experimental methods are providedbelow and elsewhere herein.

Extraction

Cytoplasmic proteins from PC12 cells, can be extracted using 40-50% ofthe CelLytic-M solution and is almost complete within 2 minutes. 100%CelLytic-M allows all cellular proteins to be extracted. The reactioncan be performed on individual cells trapped in wells or regions of amicrofluidic device. Serum can be extracted from blood using standardmethods. The cells can be spun down and this can be followed by thelysis methods described herein.

Single Cell

Spreading single cells into well array. With non-lipid membranecontaining nanopore systems, the cells can be lysed using detergents.This the protein contents of the cell can spill out. In order toencourage transport of proteins rather than nucleic acids, nucleases canbe added so that DNA and RNA are degraded, leaving only polypeptidesbeing the predominant polymer to transit through the pores. Nuclearproteins can be analysed by increasing the detergent concentration,e.g., to 100%.

Unfolding

Proteins may optionally be briefly heated to near boiling in thepresence of a reducing agent, such as dithiothreitol (DTT) or2-mercaptoethanol (beta-mercaptoethanol/BME), which further denaturesthe proteins by reducing disulfide linkages, thus overcoming some formsof tertiary protein folding, and breaking up quaternary proteinstructure (oligomeric subunits).

The following is a table shows non-limiting example of proteinbioconjugation methods (related reagents are commercially available,e.g., from Life Technologies (Carlsbad)):

-   -   Primary amines (—NH2): This group exists at the N-terminus of        each polypeptide chain (called the alpha-amine) and in the side        chain of lysine (Lys, K) residues (called the epsilon-amine).        Because of its positive charge at physiologic conditions,        primary amines are usually outward-facing (e.g., on the outer        surface) of proteins; thus, they are usually accessible for        conjugation without denaturing protein structure.    -   Carboxyls (—COOH): This group exists at the C-terminus of each        polypeptide chain and in the side chains of aspartic acid        (Asp, D) and glutamic acid (Glu, E). Like primary amines,        carboxyls are usually on the surface of protein structure.    -   Sulfhydryls (—SH): This group exists in the side chain of        cysteine (Cys, C). Often, as part of a protein's secondary or        tertiary structure, cysteines are joined together between their        side chains via disulfide bonds (—S—S—). These must be reduced        to sulfhydryls to make them available for crosslinking by most        types of reactive groups.    -   Carbonyls (—CHO): Ketone or aldehyde groups can be created in        glycoproteins by oxidizing the polysaccharide post-translational        modifications (glycosylation) with sodium meta-periodate.

The following reference is incorporated by example, and is instructiveon a number of methods for labeling specific amino acids or subsetsthereof: Angew Chem Int Ed Engl. 2009; 48(38): 6974-6998.

The following is one of the protocols for labeling amines (i.e. lysine)using Cy3 NHS Ester, Cy5 NHS ester, Alkyne STp ester, Azide NHS esterform Lumiprobe LLC (lumiprobe.com). The following protocol is accordingthe Lumiprobe recommendation.

1. Determine volume of reaction mixture. The labeling can be performedon any scale from nanomols to dozens of grams. When the scale is low,use minimal volume (10-20 uL). Higher concentrations (1-10 mg ofamino-biomolecule per mL of mixture) is optimal.

2. Dissolve NHS ester in 1/10 reaction volume of DMF or DMSO. Amine-freeDMF is preferred solvent. After the reaction, NHS ester can be stored insolution for 1-2 months at 20° C.

3. Dissolve biomolecule in 9/10 reaction volume of buffer with pH8.3-8.5. 0.1 M Sodium bicarbonate solution has appropriate pH. Otheralternatives are 0.1 M Tris buffer (although Tris has amino group, it ishindered and does not react with NHS esters), or 0.1 M phosphate buffer.Note pH is most important thing. When doing large-scale labeling(hundreds of milligrams of NHS ester), note that the mixture tends toacidify with time because of hydrolysis of NHS ester. Monitor pH, or usemore concentrated buffer then.

4. Add NHS ester solution to the solution of biomolecule, and vortexwell. Keep on ice overnight, or at room temperature during at least 4hours.

6. Purify the conjugate using appropriate method: gel-filtration formacromolecules is most universal. Precipitation and chromatography isanother alternative. Organic impurities (such as N-hydroxysuccinimide,NHS ester, acid produced by hydrolysis) are almost always easilyseparated.

For higher efficiency labelling the ratio of active ester to proteinscan be increased to as high is tolerated. A range of denaturantsincluding urea can be tested for compatibility with subsequent steps(specific concentrations are given below for labeling of cysteines).

Cysteines and Lysines can be modified quire routinely using a number ofavailable kits and protocols. But other amino acids can also bespecifically labeled. For example, it has been shown that tyrosine canbe modified through electrophilic aromatic substitutions (EAS) reactions(Stephanopoulos, N.; Francis, M. B. (2011). “Choosing an effectiveprotein bioconjugation strategy”. Nature Chemical Biology 7 (12):876-884).

Labeling of Luciferase Protein as a Model

To exemplify the approach we chose to incorporate biotinylated lysineinto combined in vitro transcription/translation (TNT kit, Promega) of aplasmid containing the Luciferin gene. The biotin was incorporated byuse of Transcend biotin lysyl-tRNA (Promega) as part of the translationreaction. The following reaction protocol was used:

TNT Quick master mix, 40 ul

Methionine, 1 ul

Plasmid DNA template (control), 2 ul

Transcend biotin lysyl-tRNA, 2 ul

Nuclease Free water, 5 ul

Total 50 ul

30 degrees C. for 90 minute

Following the reaction the biotin was reacted with streptavidin andpurified using the Slide-O-Lyser (Invitrogen).

The labeled Luciferin polypeptide was now ready for analysis.

Labeling of Titin Protein as a Model

The highly reactive Cys (SH groups) residues in Titin can be labeledusing ioadoacetamide (see Journal of Muscle Research and Cell Motility23: 499-511, 2002)

Purification

After labeling the proteins can be purified by one of a number ofavailable methods. The following is one type of kit that is available(Life Technologies) to separate protein from unlabeled reactants.

Slide-A-Lyzer™ MINI Dialysis Device, 2K MWCO, 0.1 mL, 20K MWCO, 0.1 mL(Invitrogen)

Tailing

Oligo- or poly-peptide (e.g., Polyanion) tails can be grafted onto theN- or C-terminals of proteins. There are a number of chemical approachesfor making such modifications. One example which is general toN-terminal residues, is their conversion into 2-oxoacyl groups byreaction of the α-amino group with glyoxylate, a reaction catalysed by abivalent cation, e.g., Cu2+, and a base, e.g., acetate.

An example of C-termini modification is the native chemical ligation(NCL), which is the coupling between a C-terminal thioester and aN-terminal cysteine.

Hetero-bifunctional crosslinkers or PEG can be used to make attachmentsto N- or C-terminal ends.

This allows for example, the appending of a 65-amino-acid-longglycine/serine tail including 13 interspersed negatively chargedaspartate residues. This unstructured polyanion was designed by Nivalaet al to promote capture and retention of the polypeptide end in theelectric field across the nanopore. The appended polyanion can be cappedat its C terminus with the ssrA tag, an 11-amino-acid ClpX-targetingmotif. The ssrA tag san also be added directly to the polypeptide,without the intervening glycine/serine tail.

Oligonucleotides or polynucleotides can be grafted onto the N- orC-termini of polypeptides in the same way.

Stretching Polypeptides by Molecular Combing

Polypeptides can be deposited onto a surface suitable for AFM imaging,in a chain-like substantially non-globular state, with the use ofreagents that render or maintain the chain relatively free of higherorder structures and prevent aggregation (e.g., Urea). Polypeptides canbe extended by molecular combing using a receding meniscus. This can bedone in one of two ways: (a) by moving a droplet containing thepolypeptides over a suitable surface, (b) dipping and pulling out asubstrate from a reservoir containing the polypeptides, (c) drying of adroplet on a surface.

The proteins (e.g., Titin) are diluted with PBS solution (10 mMK-phosphate pH 7.4, 140 mM NaCl, 0.02% NaN3) containing 50% glycerol toan approximate final protein concentration of 20 μg/ml. In typicalexperiments urea is added to a final concentration of 1 M to reduceprotein aggregation. Optionally 1M Guanidinium Chloride is added tominimize globular folding within the polypeptide. 20 μl sample isapplied to freshly cleaved mica and immediately spun in a rotor with13,000 RPM for 10 s. The rotor, a flat round anodized aluminum block,holds the mica sheet at a radius of 5 cm from the rotation axis of atabletop centrifuge. Following spinning, but before the complete dryingof the residual liquid layer, the mica surface is extensively washedwith distilled H2O and dried with clean N2 gas. Optionally the specimenis dried further under ambient conditions prior to AFM imaging.Optionally the sample is covered with PBS solution immediately after thecentrifugation step.

DNA Facilitated Molecular Combing.

DNA was grafted onto the N terminal of the protein (see above). TheProtein was attached to the surface via its N terminus and itsstretching was facilitated by the DNA part being stretched by a recedingmeniscus and being deposited on the surface. YOYO-1 of Sybr Goldstaining of the DNA facilitated locating of the polypeptide-DNA hybrids,allowing interrogation of the polypeptide portion to occur bysuper-resolution DNA PAINT imaging and other methods of this invention.

AFM Imaging on Mica

The purified protein (s) (Luciferin, Titin, or a proteomic mixture) wasdiluted with PBS solution (10 mM K-phosphate pH 7.4, 140 mM NaCl, 0.02%NaN3) containing 50% glycerol to an approximate final concentration of20 μg/ml. Urea and Guanidinium Chloride were each added to a finalconcentration of 1 M. 20 μl sample was applied to freshly cleaved mica(attached to a small Puck) and immediately spun by taping to the flatsection of a rotor with 13,000 RPM for 10 s. Following spinning, themica was allowed to dry.

The mica was attached via the puck to the magnetic loading surface of aMultimode Scanning Probe Microscope (Bruker, Germany). A siliconnitride, SNL-10 AFM cantilever was attached to the fluid cell of the AFMand loaded onto the AFM head. Buffer was added between the cantileverand mica surface, through one of the inlets of the fluid cell and thecantilever was brought towards the surface until the fluid formed avisible meniscus. The instrument software was opened and tapping modeselected. The laser was focused on the back of the cantilever. Thecantilever was tuned. After further approach to towards the surfaceusing the toggle on the multimode AFM, software approach was commenced.Upon tip engagement to the surface, the set point voltage was optimizedto obtain an image of sufficient quality to see labeled polypeptides onthe surface.

Optical Imaging on Mica

A TNT reaction of Luciferin was conducted but instead of incorporatingbiotin, a fluorescent dye was incorporated at the lysine residues. Theprotein was deposited on Mica as described above. The Mica wassandwiched with a cover glass with imaging buffer, containing anti-fadecomponents (e.g., SlowFade, Invitrogen). The cover glass was paced on anupright epifluorescence microscope or inverted and placed on an invertedmicroscope. Focus was obtained through the cover glass onto the Micasurface, using lamp illumination with appropriate filter for fluoresceinor 488 nm laser illumination.

Super-Resolution Imaging on Cover Glass

Polypeptides were stretched on a surface using molecular combing. A TNTreaction was conducted to incorporate biotin, and a biotinylatedoligonucleotide comprising docking sequence for DNA PAINT oligs wasattached via streptavidin. Imaging of lysine locations, closer than thediffraction limit was done by adding complementary PAINT oligos and asuper-resolution image was constructed using the DNA PAINT imagereconstruction methods (Jungmann. NanoLetters, 2010, 10: 4756-4761.

Molecular Motor Assisted Nanopore Measurements

All experiments were performed in buffer containing 200 mM KCl, 5 mMMgCl2, 10% glycerol and 25 mM HEPES-KOH pH 7.6. Setup of the nanoporedevice and insertion of an α-Hemolysin (HL) nanopore into a lipidbilayer was as follows a single α-HL nanopore was inserted into a lipidbilayer that separates two wells that each contained 100 μl of buffer. Aconstant 180 mV potential was applied across the bilayer and ioniccurrent was measured through the nanopore between Ag/AgCl electrodes inseries with an integrating patch clamp amplifier (Axopatch 200B,Molecular Devices) in voltage clamp mode. Insertion of a singlenanopores led to a current of approximately 65 pA. Data were recordedusing an analog-to-digital converter (Digidata 1440A, Molecular Devices)at 100 kHz bandwidth in whole-cell configuration then filtered at 2 kHzusing an analog low-pass Bessel filter. Experimental conditions wereprepared by the daily preparation of Buffer/ATP 5 mM and Buffer/ATP 4mM. ClpX was diluted 1:10 in Buffer/ATP 5 mM for a final concentrationof 30-100 nM ClpX6 in 4.5 mM ATP final. Then ClpX and ATP were added tothe trans compartment.

ClpX solution was used to fill the entire system before isolation of asingle α-HL nanopore. Upon insertion, the cis well was perfused with ˜6mL Buffer/ATP 4 mM. Experiments were conducted at 30° C. with 1-2 μMsubstrate added to the cis well. Similar experiments were conductedusing ClpXP in place of ClpX.

When a signal (ssrA) tag had been added to the protein(s) ClpX hexamersin the trans bath (on the opposite side of the bilayer from proteinsubstrate addition) are able to bind to the ssrA tag once it entered thetrans compartment. Translocation is facilitated in an ATP-dependentmanner.

Analysis of the current traces was used to determine blockade magnitudeduration and frequencies.

Nanopore Measurements without Molecular Motor

A nanopores set up as above was used with the following modifications.The α-HL nanopores replaced with an aerolysin nanopore, a high saltbuffer (1 M KCl and 1 M Gdm-HCl, 5 mM HEPES, pH 7.4). Data were filteredat 10 kHz and acquired at 4 μs intervals with the DigiData 1322Adigitizer coupled to Clamplex software (Axon Instruments, USA).

In addition, labeled Bovine Serum Albumin (BSA) was analysed accordingto Protein Pept Lett. 2014 March; 21(3): 256-265. To denature BSA, 1.4mg SDS (3.9 mM) with 2 mM DTT was added to the stock BSA (1 mg BSA in 1ml) solution. This mixture was heated at 450 for 5 minutes, thenimmediately cooled down in a water bath at room temperature. This BSAtreated with SDS+DTT and heated at 45°, 600 or 90° and added to the cischamber. In this experiment the center part of a solid-state nanoporedevice is a single nanopore fabricated in an insulating membrane(silicon nitride) which separates two PDMS chambers filled with saltsolution: protein samples is added to the cis chamber, and proteinmolecules move into the trans chamber after translocating through thenanopore. A pair of silver chloride electrodes is implanted in thechambers. The electrodes are used to apply a constant voltage across themembrane and to measure the ionic current through the nanopore. The cischamber is optionally electrically grounded and the trans is positivelybiased. The nanopores in silicon are fabricated by a Focused Ion Beam(FIB) or by a combination of FIB and low energy noble gas ion beam. Thenanopores are typically 10-30 nm in thickness, preferably around 16 nm,and 2-25 nm in diameter. The electrolyte solution contained 1M KCl with10 mM Tris and 1 mM EDTA at pH 7. A 1 mg/ml stock solution of BSAprotein (Sigma-Aldrich) was dissolved to make (˜15 μM) in ˜150 mM KCl TEbuffer. The trans chamber was positively biased to drive the negativelycharged BSA (pH 7) to pass through the nanopore. The ionic currentsignal was recorded using an Axopatch 200B (Molecular Devices) in eventdriven and voltage-clamp mode. The low pass Bessel filter in theAxopatch 200B was set to 10 kHz or 100 kHz.

Working Example of Cell to Analysis

Cells from NIH 3T3 adherent cell line were harvested by trypsinization,diluted with nine volumes of lysis buffer (7 M urea, 2M thiourea, 10 mMTris, 4% CHAPS, 5 mM magnesium acetate pH to 8.0) incubated on ice for30 min, and sonicated on wet ice using 25s pulses at 5-6 micronsamplitude with 1 min cooling period until clear. Centrifuged at +4° C.at 12 000×g for 10 min. Pellet discarded and protein concentration ofsupernatant determined by using an aliquot. Lysines in the proteomicsample (50 g g) were then labelled with 0.4-400 nmol of CyDye DIGE FluorCy2, Cy3 or Cy5 dye (GE Healthcare) (ideally optimized in this range).Following published results (Electrophoresis 2003, 24, 2348-2358),Cysteines were labeled by taking 25 mM proteomic sample, reduced withvariable amounts of TCEP in 8M urea, 50 mM Tris-HCl (pH 7.5 or pH 8.0)and then alkylated with fluorescent thiol-reactive dye (BODIPY TMRcadaverine IA and BODIPY F1 C 1-I) for 1.5-2 h. The reaction wastypically quenched by the addition of 150-fold excess of2-mercaptoethanol for 30 min. In some cases, labeled proteins werepurified from free dye using a PD-10 column. In general the dyeconcentration must be equal to or greater than TCEP concentration. Atypical ratio of, 9:1 for TCEP over thiol and, 1.125:1 of dye over TCEP(or 10:1 of dye over thiol) was effective in reducing and labelingproteins that had multiple disulfide bonds in a 90 min reaction time,complete labeling was achieved at a dye:thiol ratio of 10:1 and aTCEP:thiol ratio of 9:1 (but can be increased to 75:1) efficientlabeling takes place even in the presence of 8M urea; this kept thepolypeptides in an unfolded state. The labeled proteins were thenoptionally purified or directly analysed by nanopores analysis, withpost-processing filters applied to remove noise from unpurified reactioncomponents such as unreacted dyes.

Single cells are processed in the same way by conducting lysis in amicrowell (10-30 pL volume) (after Sasuga et al. Anl. Chem. 2008, 80,9141-9149) and labeling directly (without concentration being determinedor cell debris being removed). In some experiments these wells containnanopores systems as described elsewhere in this document, buffer andreagents and appropriately localized electrodes, for nanopores analysisof the sites of labelling on individual proteins to be conducted.Alternatively, the protocol is carried out in a microfluidic device inwhich individuals cells are trapped and the contents released into amicro-channel or chamber, using the above lysis buffer, and collectingthe proteomic mixture in a region of the chip where the labelingreagents were added. The proteomic mixture is then fed into a nanoporesmeasuring system or into nanochannels or attached to a surface withinthe chip and stretched, allowing optical imaging of the location of thelabels along the polypeptides, to be conducted.

Finding a Match Between Experimental and in Silico Data

The coordinates of the labels along each polypeptide were determinedusing image processing tools (ImageJ) and stored in computer memory. Theexperimentally derived coordinates from the model proteins were thencomputationally compared to coordinates of the protein stored incomputer memory. In the case when data from a mixture of proteins ofunknown composition was obtained experimentally, the labeling pattern oneach polypeptide ass compared to a the expected pattern of a list ofproteins in the database (e.g., using Amazon (EC2/S3) and Digital Ocean)to find the best match.

While several embodiments of the present disclosure have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the presentdisclosure. More generally, those skilled in the art will readilyappreciate that all parameters, dimensions, materials, andconfigurations described herein are meant to be exemplary and that theactual parameters, dimensions, materials, and/or configurations willdepend upon the specific application or applications for which theteachings of the present disclosure is/are used. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of thedisclosure described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, thedisclosure may be practiced otherwise than as specifically described andclaimed. The present disclosure is directed to each individual feature,system, article, material, and/or method described herein. In addition,any combination of two or more such features, systems, articles,materials, and/or methods, if such features, systems, articles,materials, and/or methods are not mutually inconsistent, is includedwithin the scope of the present disclosure.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

It should be appreciated that features of separately-recited embodimentscan be combined in any desired combination which may be apparent tothose skilled in the art.

1. A method for analyzing a protein, the method comprising: i. unfoldinga protein into a polypeptide; ii. tagging said polypeptide with reagentsthat recognize specific residues on said polypeptide; iii. rendering thepolypeptide such that the distance between tag sites along thepolypeptide is determined via the time elapsed between detection oftags; and iv. detecting the tags.
 2. The method of claim 1, wherein stepiii is performed before step ii.
 3. The method of claim 1, wherein stepii is performed before step iii.
 4. The method according to claim 1where the unfolding is conducted via a, mechanical, chemical orenzymatic method or a combination thereof.
 5. The method according toclaim 1 where the unfolding is facilitated by a molecular motorcomprising ClpX, ClpXP 6-9. (canceled)
 10. The method according to claim1, wherein the tagging is conducted by chemical or biological means. 11.The method according to claim 10, wherein the tagging is performedusing, NHS ester, malemide, or nickel-histidine chemistry.
 12. Themethod according to claim 10 where the tagging is performed usingantibodies, affybodies, or aptamers.
 13. The method according to claim10, wherein the tagging comprises associating the polypeptide with adetectable label.
 14. The method according to claim 13, whereindetectable label comprises a fluorescent/luminescent label, a lightscattering label, a contrast label, size label or a nanoparticle label.15. A method for analyzing a protein, the method comprising: i.unfolding a protein into a polypeptide ii. rendering the polypeptidesuch that the location, distance between and/or order of sites of choicealong the polypeptide is resolvable by detecting features of thepolypeptide along its length; and iii. detecting features of thepolypeptide along the polypeptide length.
 16. The method according toclaim 1, wherein the polypeptide is passed linearly through anano-constriction/gap or nanopore.
 17. The method according to claim 1,wherein the features along the length are detected by perturbation ofthe interaction of two or more entities comprising FRET, RET, electrontunneling/transfer donor and acceptor or semi-conductor source anddrain. 18-19. (canceled)
 20. The method according to claim 15, whereinthe tag sites along the polypeptide are resolved by nanopore/nanogapdetection. 21-23. (canceled)
 24. The method according to claim 1,wherein the specific residues are detected by optical microscopy, Ramanspectroscopy, SERS/SERRS, lens-free microscopy, X-ray microscopy,electron microscopy, scanning probe microscopy, nanopore/nanogapdetection, or near-field optical detection. 25-39. (canceled)
 40. Themethod according to claim 1, wherein the polypeptide is fused with amolecule, not comprising amino acids, prior to rendering the polypeptidesuch that the distance between tag sites along the polypeptide isdetermined via the time elapsed between detection of tags. 41-42.(canceled)
 43. A method according to claim 40, wherein the molecule, notcomprising amino acids, comprises a cholesterol. 44-47. (canceled)
 48. Adevice for carrying out the method according to 1, the device comprisinga macro interface, a microscale portion configured for containingligands or for receiving ligands from an external reservoir, and ananoscale portion configured for detecting or analyzing linearpolypeptides. 49-53. (canceled)
 54. The method according to claim 15,wherein the polypeptide is passed linearly through anano-constriction/gap or nanopore.
 55. The method according to claim 15,wherein the features along the length are detected by perturbation ofthe interaction of two or more entities comprising FRET, RET, electrontunneling/transfer donor and acceptor or semi-conductor source anddrain.